| Historically,transcription pre-initiation complex(PIC)assembling was recognized as a critical step in eukaryotic transcriptional regulation catalyzed by RNA polymerase II(also known as Pol II or RNAP II).Studies in recent years have revealed that the release of promoter-proximal paused Pol II was another rate-limiting step in transcription.Paused Pol II is highlighted by the existence of many negative factors that repress transcription elongation,such as DSIF,NELF,TRIM28 and epigenetic regulation factors including Polycomb Repressive Complexes 1(PRC1).However,the recruiting mechanism to target genes and the molecular coordination among these factors are not clear.In addition,Gdown1 is a newly discovered protein involved in transcription elongation control,and the function of Gdown1 and factors interact with Gdown1 remain elusive.Nonetheless,a recent study demonstrated that Drosophila Spt5,a subunit of DSIF,interacted both physically and genetically with the PRC1 Pleiohomeotic(Pho)in S2 cells,while the homologous interaction between PRC1 and DSIF in mammals has not been reported.IP experiments and mass spectrometry analyses of prostate cancer cell lines showed that Spt5 was one of the proteins interacted with Ring1 B,a core component of PRC1,while no further experimental evidence was performed to confirm this interaction.TRIM28,a co-repressor with novel functions as a transcription elongation factor,was actually found earlier in our study to interplay with PRC1.For further understand the mechanism of transcription elongation control,the aims of this thesis include two aspects: 1)Explore the molecular functions of Gdown1;2)Characterize the molecular interplays among negative regulators in transcription elongation control.Methods:(1)Test the expression of Gdown1 splicing variants in human and mouse through real-time PCR.(2)Generate stable cell lines that ectopic express Gdown1 or with Gdown1 conditionally knocked down with a lentiviral expression system.(3)Characterize subcellular localization information of both endogenous and exogenous Gdown1 through cell extraction/ immunoblotting assays and fluorescence observation,respectively.(4)Employ BiFC and GST pull-down assays to determine the interaction among PRC1 components(Ring1B,Cbx2,Cbx7)and negative elongation factors [Gdown1,TRIM28,DSIF(composed by Spt5 and Spt4)].Results:(1)The full length form of mouse Gdown1(366 aa),expresses in a much higher level comparing to another expressed splicing variant(262 aa)in various mouse cell lines and tissues.On contrast,the human counterpart of full length Gdown1(368 aa)is the only expressed isoform in HEK293 T and HeLa cells.(2)A series of stable cell lines with the expression levels of Gdown1 manipulated(either overexpressed or knocked down)were generated and verified.(3)The endogenous Gdown1 is mainly located in the nucleus in HEK293 T cells while ectopically expressed Gdown1-Venus-Flag is mainly exhibiting fluorescent signals in the cytoplasm.(4)A series of novel interactions are uncovered.First,Ring1 B directly interacts to Spt5 in human.Second,the two subunits of DSIF both interacted with TRIM28.Finally,the self interaction between Gdown1 molecules and the interaction between Gdown1 and Spt4 are very dramatically detected in BiFC assays.Conclusions: The Gdown1 expression information and series stable cell lines generated in this thesis would be very helpful for future functional studies of Gdown1 in transcription elongation control.Our data provide strong evidence that distinct negative elongation factors and epigenetic regulatory factors may work synergistically as a whole in stabilizing poised Pol II elongation complex and achieve the transcription repression effect.The novel interactions between PRC1 and negative elongation factors organically combined the epigenetic regulators and classic transcription elongation regulators in a big picture,therefore significantly expanded our understanding of transcription elongation control. |
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